The goal of the proposed research is to determine the mechanism by which the amino acid sequence of a protein directs the rapid and efficient folding to the unique native conformation. Given that all the required information is contained in the amino acid sequence, a collection of mutations will be constructed in a pair of proteins from Escherichia coli using genetic engineering techniques. The effects of these replacements on the stability and kinetics of folding of the alpha subunit of tryptophan synthase and the trp aporepressor will be monitored by optical spectroscopy. The perturbations on the equilibrium constant and various rate constants will be analyzed in terms of reaction coordinate diagrams which emphasize the relative energies of various species and transition states in the folding reaction. The results will be interpreted in terms of kinetic models of folding which have been developed for both proteins, and the existing X-ray structures. The alpha subunit is an interesting target because it has an alpha/beta barrel structure which unfolds via a stable intermediate. Mutations are planned in the beta strand core and the alpha helix periphery which will probe the structure of both early and late folding intermediates and the transition state of the rate limiting step in folding. Multiple replacements will test barrel architecture. The trp aporepressor is an interdigitated dimer whose folding mechanism will provide insight into the formation of quaternary structure. The information obtained will be useful in the prediction of tertiary structure from primary sequence, in understanding the molecular basis of inherited diseases and in the design of new enzyme catalysts.